Lipoproteins are globular, micelle-like particles that consist of a non-polar core of acylglycerols and cholesteryl esters surrounded by an amphiphilic coating of protein, phospholipid and cholesterol. Lipoproteins have been classified into five broad categories on the basis of their functional and physical properties: chylomicrons, very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density lipoproteins (LDL), and high density lipoproteins (HDL). Chylomicrons transport dietary lipids from intestine to tissues. VLDLs, IDLs and LDLs all transport triacylglycerols and cholesterol from the liver to tissues. HDLs transport endogenous cholesterol from tissues to the liver
Lipoprotein particles undergo continuous metabolic processing and have variable properties and compositions. Lipoprotein densities increase without increasing particle diameter because the density of their outer coatings is less than that of the inner core. The protein components of lipoproteins are known as apolipoproteins. At least nine apolipoproteins are distributed in significant amounts among the various human lipoproteins.
Apolipoprotein C-III (ApoCIII) is a constituent of HDL and of triglyceride (TG)-rich lipoproteins. Elevated ApoCIII is associated with hypertriglyceridemia. Accordingly, ApoCIII has a role in hypertriglyceridemia, a risk factor for coronary artery disease (Davidsson et al., J. Lipid Res. 2005. 46: 1999-2006). ApoCIII slows clearance of triglyceride-rich lipoproteins by inhibiting lipolysis, both through inhibition of lipoprotein lipase and by interfering with lipoprotein binding to cell-surface glycosaminoglycan matrix (Shachter, Curr. Opin. Lipidol., 2001, 12, 297-304).
The gene encoding human apolipoprotein C-III (also called APOC3, APOC-III, ApoCIII, and APO C-III) was cloned in 1984 by three research groups (Levy-Wilson et al., DNA, 1984, 3, 359-364; Protter et al., DNA, 1984, 3, 449-456; Sharpe et al., Nucleic Acids Res., 1984, 12, 3917-3932). The coding sequence is interrupted by three introns (Protter et al., DNA, 1984, 3, 449-456). The human ApoCIII gene is located approximately 2.6 kb to the 3′ direction of the apolipoprotein A-1 gene and these two genes are convergently transcribed (Karathanasis, Proc. Natl. Acad. Sci. U.S.A., 1985, 82, 6374-6378). Also cloned was a variant of human apolipoprotein C-III with a Thr74 to Ala74 mutation from a patient with unusually high level of serum apolipoprotein C-III. As the Thr74 is O-glycosylated, the Ala74 mutant therefore resulted in increased levels of serum ApoCIII lacking the carbohydrate moiety (Maeda et al., J. Lipid Res., 1987, 28, 1405-1409). Other variants or polymorphisms that modulated Apo CIII expression were later identified. Some of the polymorphsims elevated ApoCIII. Elevated ApoCIII levels were associated with elevated triglyceride (TG) levels and diseases such as cardiovascular disease, metabolic syndrome, obesity and diabetes (Chan et al., Int J Clin Pract, 2008, 62:799-809; Onat et al., Atherosclerosis, 2003, 168:81-89; Mendivil et al., Circulation, 2011, 124:2065-2072).
Five polymorphisms have been identified in the promoter region of the gene: C (at position −641 of the gene) to A, G (at position −630 of the gene) to A, T (at position −625 of the gene) to deletion, C (at position −482 of the gene) to T and T (at position −455 of the gene) to C. All of these polymorphisms are in linkage disequilibrium with the SstI polymorphism in the 3′ untranslated region. The SstI polymorphic site distinguishes the S1 and S2 alleles and the S2 allele has been associated with elevated plasma triglyceride levels (Dammerman et al., Proc. Natl. Acad. Sci. U.S.A., 1993, 90, 4562-4566). The ApoCIII promoter is downregulated by insulin and this polymorphic site abolishes the insulin regulation. Thus the potential overexpression of ApoCIII resulting from the loss of insulin regulation may be a contributing factor to the development of hypertriglyceridemia associated with the S2 allele (Li et al., J. Clin. Invest., 1995, 96, 2601-2605). The T (at position −455 of the gene) to C polymorphism has been associated with an increased risk of coronary artery disease (Olivieri et al., J. Lipid Res., 2002, 43, 1450-1457). Other polymorphisms in the human ApoCIII gene that have been associated with elevated ApoCIII and/or triglyceride expression include: C (at position 1100) to T, C (at position 3175) to G, T (at position 3206) to G, C (at positions 3238) to G, etc. (Tilly et al., J. Lipid Res., 2003, 44:430-436; Waterworth et al., Arterioscler Thromb Vasc Biol, 2000, 20:2663-2669; Petersen et al., N Engl J Med, 2010, 362:1082-1089).
In addition to insulin, other regulators of ApoCIII gene expression have been identified. A response element for the nuclear orphan receptor rev-erb alpha has been located at positions −23 to −18 of the gene, in the ApoCIII promoter region. Rev-erb alpha decreases ApoCIII promoter activity (Raspe et al., J Lipid Res., 2002, 43, 2172-2179). The ApoCIII promoter region from positions −86 to −74 of the gene is recognized by two nuclear factors CIIIb1 and CIIIB2 (Ogami et al., J. Biol. Chem., 1991, 266, 9640-9646). ApoCIII expression is also upregulated by retinoids acting via the retinoid X receptor, and alterations in retinoid X receptor abundance affects ApoCIII transcription (Vu-Dac et al., J. Clin. Invest., 1998, 102, 625-632). Specificity protein 1 (Sp1) and hepatocyte nuclear factor-4 (HNF-4) have been shown to work synergistically to transactivate the apolipoprotein C-III promoter via the HNF-4 binding site (Kardassis et al., Biochemistry, 2002, 41, 1217-1228). HNF-4 also works in conjunction with SMAD3-SMAD4 to transactivate the ApoCIII promoter (Kardassis et al., J Biol. Chem., 2000, 275, 41405-41414).
Transgenic and knockout mice have further defined the role of ApoCIII in lipolysis. Overexpression of ApoCIII in transgenic mice leads to hypertriglyceridemia and impaired clearance of VLDL-triglycerides (de Silva et al., J. Biol. Chem., 1994, 269, 2324-2335; Ito et al., Science, 1990, 249, 790-793). Knockout mice with a total absence of the ApoCIII protein exhibited significantly reduced plasma cholesterol and triglyceride levels compared with wild-type mice and were protected from postprandial hypertriglyceridemia (Maeda et al., J. Biol. Chem., 1994, 269, 23610-23616).
Total plasma ApoCIII levels have been identified as a major determinant of serum triglycerides, and epidemiological studies have demonstrated that ApoCIII and ApoB lipoproteins that have ApoCIII as a component independently predict coronary heart disease (Sacks et al., Circulation. 2000. 102: 1886-1892; Lee et al., Arterioscler Thromb Vasc Biol. 2003. 23: 853-858). Studies also demonstrate that ApoCIII is a key determinant in the clearance of triglyceride-rich lipoproteins and its remnants in hypertriglyceridaemic states, including visceral obesity, insulin resistance and the metabolic syndrome (Mauger et al., J. Lipid Res. 2006. 47: 1212-1218; Chan et al., Clin. Chem. 2002. 278-283; Ooi et al., Clin. Sci. 2008. 114: 611-624).
Hypertriglyceridemia is a common clinical trait associated with an increased risk of cardiometabolic disease (Hegele et al. 2009, Hum Mol Genet, 18: 4189-4194; Hegele and Pollex 2009, Mol Cell Biochem, 326: 35-43) as well as of occurrence of acute pancreatitis in the most severe forms (Toskes 1990, Gastroenterol Clin North Am, 19: 783-791; Gaudet et al. 2010, Atherosclerosis Supplements, 11: 55-60; Catapano et al. 2011, Atherosclerosis, 217S: S1-S44; Tremblay et al. 2011, J Clin Lipidol, 5: 37-44). Examples of cardiometabolic disease include, but are not limited to, diabetes, metabolic syndrome/insulin resistance, and genetic disorders such as familial chylomicronemia, familial combined hyperlipidemia and familial hypertriglyceridemia.
Hypertriglyceridemia is the consequence of increased production and/or reduced or delayed catabolism of triglyceride (TG)-rich lipoproteins: VLDL and, to a lesser extent, chylomicrons (CM). Borderline high TGs (150-199 mg/dL) are commonly found in the general population and are a common component of the metabolic syndrome/insulin resistance states. The same is true for high TGs (200-499 mg/dL) except that as plasma TG levels increase, underlying genetic factors play an increasingly important etiologic role. Very high TGs (≧500 mg/dL) are most often associated with elevated CM levels as well, and are accompanied by increasing risk for acute pancreatitis. The risk of pancreatitis is considered clinically significant if TG exceeds 880 mg/dL (>10 mmol) and the European Atherosclerosis Society/European Society of Cardiology (EAS/ESC) 2011 guidelines state that actions to prevent acute pancreatitis are mandatory (Catapano et al. 2011, Atherosclerosis, 217S: S1-S44). According to the EAS/ESC 2011 guidelines, hypertriglyceridemia is the cause of approximately 10% of all cases of pancreatitis, and development of pancreatitis can occur at TG levels between 440-880 mg/dL. Based on evidence from clinical studies demonstrating that elevated TG levels are an independent risk factor for atherosclerotic CVD, the guidelines from both the National Cholesterol Education Program Adult Treatment Panel III (NCEP 2002, Circulation, 106: 3143-421) and the American Diabetes Association (ADA 2008, Diabetes Care, 31: S12-S54.) recommend a target TG level of less than 150 mg/dL to reduce cardiovascular risk.
ApoCIII-knockout mice had normal intestinal lipid absorption and hepatic VLDL triacylglycerol secretion, but a rapid clearance of VLDL triacylglycerols and VLDL cholesteryl esters from plasma that may explain the observed hypolipidaemia (Gerritsen et al., J. Lipid Res. 2005. 46: 1466-1473; Jong et al., J. Lipid Res. 2001. 42: 1578-1585). VLDL particles with ApoCIII have been cited to play a major role in identifying the high risk of coronary heart disease in hypertriglyceridemia (Campos et al., J. Lipid Res. 2001. 42: 1239-1249). A genome-wide association study found a naturally occurring ApoCIII null mutation in Lancaster Amish people demonstrated a favorable lipid profile and apparent cardioprotection, with no obvious detrimental effects (Pollin et al., Science. 2008. 322: 1702-1705). The mutation carriers are observed to have lower fasting and postprandial serum triglycerides and LDL-cholesterol, and higher levels of HDL-cholesterol.
The HDL class of lipoproteins comprises a heterogeneous and polydisperse population of particles that are the most dense and smallest of size (Havel and Kane. In, The Metabolic & Molecular Bases of Inherited Disease. 8th Edition. McGraw-Hill, New York, 2001:2705-16). HDL is a macromolecular complex of lipids (cholesterol, triglycerides and phospholipids) and proteins (apolipoproteins (apo) and enzymes). The surface of HDL contains chiefly apolipoproteins A, C and E. The function of some of these apoproteins is to direct HDL from the peripheral tissues to the liver. Serum HDL levels can be affected by underlying genetic causes (Weissglas-Volkov and Pajukanta, J Lipid Res, 2010, 51:2032-2057)
Epidemiological studies have indicated that increased levels of HDL protect against cardiovascular disease or coronary heart disease (Gordon et al., Am. J. Med. 1977. 62: 707-714). These effects of HDL-cholesterol are independent of triglyceride and LDL-cholesterol concentrations. In clinical practice, a low plasma HDL-cholesterol is more commonly associated with other disorders that increase plasma triglycerides, for example, central obesity, insulin resistance, type 2 diabetes mellitus and renal disease (chronic renal failure or nephrotic proteinuria) (Kashyap. Am. J. Cardiol. 1998. 82: 42U-48U).
Currently, there are no known direct therapeutic agents that affect the function of ApoCIII. The hypolipidemic effect of the fibrate class of drugs has been postulated to occur via a mechanism where peroxisome proliferator activated receptor (PPAR) mediates the displacement of HNF-4 from the apolipoprotein C-III promoter, resulting in transcriptional suppression of apolipoprotein C-III (Hertz et al., J. Biol. Chem., 1995, 270, 13470-13475). The statin class of hypolipidemic drugs also lower triglyceride levels via an unknown mechanism, which results in increases in lipoprotein lipase mRNA and a decrease in plasma levels of apolipoprotein C-III (Schoonjans et al., FEBS Lett., 1999, 452, 160-164). Consequently, there remains a long felt need for additional agents capable of effectively inhibiting apolipoprotein C-III function.
Antisense technology is emerging as an effective means for reducing the expression of certain gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of ApoCIII.
We have previously disclosed compositions and method for inhibiting ApoCIII by antisense compounds in US 20040208856 (U.S. Pat. No. 7,598,227), US 20060264395 (U.S. Pat. No. 7,750,141), and WO 2004/093783. In the present application, we disclose the unexpected result that antisense inhibition of ApoCIII resulted in the elevation of HDL levels and decrease in postprandial triglyceride levels. This result will be useful, for example, to treat, prevent, delay, decrease or ameliorate any one or more diseases, such as cardiovascular disease (e.g., coronary heart disease or atherogenic diseases). For example, elevated postprandial (non-fasting) triglyceride levels have been identified as a significant risk factor for cardiovascular diseases (Bansal et al., JAMA, 2007, 298:309-16; Nordestgaard et al., JAMA, 2007, 298:299-308), Also, in the present application, inhibition of ApoCIII expression unexpectedly results in increased chylomicron clearance and is therefore important in the prevention of chylomicronemia (Chait et al., 1992, Adv Intern Med. 1992, 37:249-73), a dyslipidemic state caused by improper clearance of chylomicron triglyceride. Severe forms of chylomicronemia can lead to pancreatitis, a life-threatening condition. By inhibiting intestinal ApoCIII, inhibition of lipoprotein lipase would be reduced, and chylomicron triglyceride clearance would be enhanced, thereby preventing pancreatitis.